Quantifiler® Trio Kit and forensic samples management: A matter of degradation

doi:10.1016/j.fsigen.2014.12.005

Forensic Science International: Genetics

Volume 16, May 2015, Pages 77-85

https://doi.org/10.1016/j.fsigen.2014.12.005 Get rights and content

Highlights

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We test the effectiveness of Quantifiler^® Trio Kit in detecting DNA degradation.
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The Quantifiler^® Trio Kit is evaluated in combination with Globalfiler^® Kit.
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The Degradation Index is evaluated by studying the peak height decay in the profile.
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Typing performance was evaluated for different PCR protocols and DI categories.
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Operative guidelines are proposed to differentiate PCR strategy at increasing of DI.

Abstract

DNA collected from crime scenes may have experienced different levels of degradation. This is mainly due to sample exposure to different environmental factors. The impact of DNA degradation on short tandem repeat (STR) profiling can lead to partial or null information and in some cases, the identification of the trace may fail. The availability of a system enabling the assessment not only of the quantity of the DNA but also of its quality in terms of degradation would result in shorter time for sample processing, more reliable identifications and cost reduction by predicting the quality of the DNA profiles prior to STR analysis. We report here a study on 181 selected degraded DNA samples extracted from real crime scene evidence. The selected samples were processed by combining the use of a new commercial quantification kit (Quantifiler^® Trio) with a new 24 marker multiplex PCR amplification kit (Globalfiler^® Kit). Applying different statistical analyses we investigated the reliability of the Degradation Index provided by the Quantifiler^® Trio in determining the level of DNA degradation in a forensic sample. This useful information can be used to predict the quality of the profile obtained after STR amplification. The combination of such a quantification kit with different PCR protocols allowed us to define practical guidelines for processing degraded forensic DNA samples with a simplified and comprehensive approach.

Introduction

The analysis of degraded DNA is one of the main challenges in the forensic laboratory, and it is also one of the most common situations encountered when dealing with DNA that has been exposed to environmental and microbial factors. Degradation tends to affect the higher molecular weight markers preferentially and leads to a significant increase in allele drop-out [1]. A statistical model to calculate allelic drop-out probabilities in degraded samples enabling improved interpretation of genetic data has been recently proposed [2], [3].

Degraded samples have been approached in several different ways in order to maximize the amount of genotyping information. One of the most common approaches is the use of mini-STRs generating amplicons of less than 220 bp. This approach may help in amplifying even severely degraded DNA [4], [5]. The combined use of different commercial STR kits amplifying the same markers may also help to reach a sufficient power of discrimination on degraded samples especially when mini-STRs are targeted in these kits [6], [7]. When the samples are not only degraded but also severely inhibited or contain low concentrations of DNA, PCR amplification replicates are strongly recommended [8]. This may help in confirming the genotypes but at the same time leads to DNA consumption. When dealing with LT (low template) DNA, the total amount of DNA available may not be enough for replicates occasionally rendering the analysis inconclusive. In addition, the use of different kits for amplifying the same DNA has a direct impact on the cost per reaction which can have a significant impact for laboratories that process thousands of samples per year.

The GlobalFiler^® PCR Amplification Kit a 6-dye, short tandem repeat (STR) multiplex assay which combines the 13 original CODIS loci with the expanded European Standard Set including 7 additional loci and the highly discriminating SE33 locus, has been recently developed and introduced in the market [9]. This kit also includes a total of 10 mini STR amplicons (<220 bp) that could allow the amplification of a sufficient number of markers for identification purposes even when the sample is severely compromised in terms of degradation.

One of the most discussed and critical steps in the casework DNA analysis workflow is the assessment of the quality and quantity of the DNA. The major limits of the commercially available quantification kits were mainly the lack of an effective and realistic degradation assessment and insufficient sensitivity [10]. During the last 5 years the STR multiplex amplification kits have been improved in terms of sensitivity and robustness to PCR inhibitors, and it is now possible to generate full profiles with amounts of DNA template as low as 30 pg [6]. Partial, yet still informative, profiles can also be obtained with lower amounts of DNA.

The need for a more sensitive solution, closing the gap between the STR multiplex and the quantification kit sensitivity, in addition to the declared ability to detect the level of degradation in a DNA sample, led us to test the new Quantifiler^® Trio DNA Quantification Kit [11], [12]. Custom homebrew solutions giving similar information about the level of degradation [13] have been published, but were not considered for an ISO/IEC 17025 environment like ours.

The Quantifiler^® Trio Kit detects two different human multi-copy autosomal targets producing a small amplicon of 80 bp (SA – small autosomal target) and a larger amplicon of 214 bp (LA – large autosomal target) that aid in assessing DNA concentration and the level of degradation in a sample.

The main intent of the study was to evaluate the reliability of the new Quantifiler^® Trio Kit in providing information as to the level of the DNA degradation in typical forensic samples and to predict the quality of profiles obtained after genotyping. With this purpose a total of 181 forensic samples from adjudicated casework (oral swab, blood, touch DNA, bone, sperm and hair) were quantified using the new Quantifiler^® Trio Kit. The level of DNA degradation in the samples was assessed by examining the ratio between the concentration of small and large autosomal amplicons (Degradation Index or DI) [11]. A total of 95 out of 181 samples chosen for having sufficient DNA available in terms of concentration and volume were amplified with the Globalfiler^® PCR Amplification Kit.

The profiles obtained after STR-amplification were analyzed in terms of RFU signal and fragment length in order to correlate “the quality” of the entire profile to the DI parameter obtained in quantification. Furthermore, we examined the level of degradation (DI) against the number of markers typed with the Globalfiler^® Kit.

In order to define some operative and practical guidelines for the combined use of the Quantifiler^® Trio and Globalfiler^® Kits for routine analysis, some of the samples previously amplified using 0.5 ng of input DNA were re-amplified using different protocols. The samples amplified with both Globalfiler^® and NGM SElect™ PCR Amplification Kits were used to calculate the average Random Match Probability (RMP) with the aim of showing which is the most informative kit according to the profile quality.

Finally, in order to optimize the typing process from a workflow perspective, saving money and precious forensic samples for possible further and future analysis, we propose some practical considerations for managing degraded samples in routine casework.

Section snippets

Samples

A set of 181 forensic samples from adjudicated casework (oral swab, blood, touch DNA, bone, sperm, hair) were selected on the basis of the profile previously obtained. Profiles with different degrees of degradation as well as single source profiles and mixtures with a distinguishable major contributor were selected. The level of degradation was assessed by visual screening of the profiles obtained with different commercial amplification kits, considering the ratio between the approximate peak

Results and discussion

In order to study the practical impact of the new Quantifiler^® Trio Kit on our workflow, a set of 181 evidence samples from adjudicated casework (oral swabs, blood, touch DNA, bones and human remains, sperm and hair) were selected on the basis of the DNA profile obtained during the first analysis. Only single profiles and mixtures with a distinguishable major contributor were selected with the aim of including a range of degrees of DNA degradation.

Conclusion

Several samples selected in this study were demonstrated to be challenging and time consuming using our existing protocols, requiring PCR input adjustment and multiple amplifications to obtain a useful profile. The results in this study demonstrated that the DI parameter information, provided by the Quantifiler^® Trio Kit, is effective in characterizing degraded samples. The information given by the DI could be used to predict the quality of the expected profile and to decide how to process the

Acknowledgments

The authors would like to thank Gabriel Jacquelyn for useful discussion. The authors also express gratitude to Thermo Fisher Scientific for supplying the prototype Quantifiler^® Trio Kit used in this study.

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Citation Excerpt :
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¹: The authors equally contributed to this paper.

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Quantifiler® Trio Kit and forensic samples management: A matter of degradation

Highlights

Abstract

Introduction

Section snippets

Samples

Results and discussion

Conclusion

Acknowledgments

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet. Supp. Ser.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Legal Med.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

A study of the effects of degradation and template concentration on the amplification efficiency of the miniplex primer sets

J. Forensic Sci.

The development of reduced size STR amplicons as tools for analysis of degraded DNA

J. Forensic Sci.

Development and validation of the AmpFℓSTR® MiniFiler™ PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

J. Forensic Sci.

Quantifiler^® Trio Kit and forensic samples management: A matter of degradation

Development and validation of the AmpFℓSTR^® MiniFiler™ PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA